Methicillin-resistant Staphylococcus aureus (MRSA) is a prevalent and rapidly growing nosocomial pathogen problem. Not only has the incidence of MRSA among hospital S. aureus isolates reached 50% (Am. J. Infect. Control 27: 520–532), there is also an emerging prevalence of MRSA strains in the community (Chambers, et al., Emerging Infectious Diseases 7: 178–182). The resistance of MRSA to methicillin and other β-lactam antibiotics is mediated by the acquired penicillin-binding protein 2a (PBP2a). PBP2a is a transpeptidase involved in cell wall peptidoglycan biosynthesis and has very low affinity for these antibiotics. A rational approach to anti-MRSA drug development is to restore sensitivity to R-lactam antibiotics by directly targeting this molecular mechanism of resistance.
Evolutionary Chemistry™ is a methodology for the discovery of small molecule pharmaceutical lead compounds. Evolutionary Chemistry™ is unique in that it integrates the steps of small molecule synthesis and high throughput screening into a single system. This is accomplished by utilizing the ability of RNA to catalyze chemical transformations that can create drug-like molecules, and exploiting this ability to assemble an enormous small molecule library. By incubating a large library of reactant-coupled, random-sequence, modified RNAs (approximately 1015 unique potential biocatalysts) with a library of small molecule reactants (104–106 unique constituents), a library of 105–108 potential lead compounds can be generated. Potential lead compounds remain attached to the biocatalysts responsible for their formation and are thus addressable. The biocatalyst-assembled product library is then subjected to evolutionary pressures that demand that the selected small molecules have specified properties (such as high affinity for a drug target). Biocatalysts conjugated to lead compounds that exhibit the demanded properties are enzymatically amplified. The biocatalyst sequence-specific small molecule assembly is reliably reproduced in subsequent cycles of biocatalysis, selection, and amplification. These cycles are iterated with increasing evolutionary pressure until the most effective lead compounds evolve from the population.
In the most general embodiments, a nucleic acid-reactant test mixture is formed by attaching a first reactant to each of the nucleic acids in a test mixture (containing 102 to 1018 nucleic acids with randomized sequences). The nucleic acid-reactant test mixture is treated with other free reactants that will combine with the first reactant to form different products. It is important to note that from the nucleic acid test mixture, discrete nucleic acid sequences will be associated with facilitating the formation of the different shaped products. The products may differ in shape, reactivity or both shape and reactivity. Partitioning of the desirable product shape or reactivity is accomplished by binding to or reaction with a target. Proteins, small molecules, lipids, saccarides, etc., are all examples of targets. After binding to or reacting with the target the non-interacting products, are partitioned from the interacting products, and discarded. The nucleic acid associated with the interacting product is then amplified by a variety of methods known to those experienced in the art. This nucleic acid is then used to facilitate the assembly of the desirable product by facilitating the specific reaction to form the selected product on treatment with the mixture of starting reactants. In a typical reaction, the amplified nucleic acid can be reattached to the first reactant, however, said reattachment is not always required. This is an idealized case and in many examples the nucleic acid facilitator may assemble more than one product from the starting mixture, but all of the products selected will have the desired properties of binding to or chemical reaction with the target.
The overall process is described in more detail in, for example, U.S. Pat. Nos. 6,048,698; 6,030,776; 5,858,660;5,789,160; 4 5,723,592; and 5,723,289, each of which is entitled “Parallel SELEX,” and each of which is incorporated herein by reference in its entirety. These patents are hereinafter referred to collectively as the “Parallel SELEX patents.”
The present invention provides novel monobactams with anti-PBP2a activity that were identified using the aforementioned Evolutionary Chemistry™ process.